瓜实蝇热激蛋白Hsp90基因克隆及表达分析
Cloning and expression of the Bactrocera cucurbitae (Coquillett) heat shock protein 90 gene
姜建军** 黄立飞 黎柳锋 陈红松 王凤英** 杨 朗***
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DOI：10.7679/j.issn.2095-1353.2019.052
作者单位：（广西壮族自治区农业科学院植物保护研究所，广西作物病虫害生物学重点实验室，南宁530007）
中文关键词：瓜实蝇；高温胁迫；抗药性；Hsp90；表达
英文关键词：Bactrocera cucurbitae; heat stress; resistance; Hsp90; gene expression
中文摘要：【目的】 热激蛋白基因Hsps在生物体抗逆性过程中具有重要的作用，本文旨在通过阐释瓜实蝇Bactrocera cucurbitae (Coquillett)热激蛋白基因Hsp90在其对环境温度适应性及抗药性过程中的功能作用，为瓜实蝇综合治理提供理论基础。【方法】 采用RACE-PCR技术克隆瓜实蝇的Hsp90基因cDNA序列，利用生物信息学工具分析序列特征，并通过实时定量PCR技术分析高温胁迫、阿维菌素诱导和抗性及敏感品系中该基因的表达情况。【结果】 克隆获得的瓜实蝇Hsp90基因cDNA全长2 654 bp，命名为Bc-Hsp90，GenBank登录号为KP864677，含2 145 bp的开放阅读框，编码715个氨基酸蛋白，具有HSP90蛋白家族共有模式和1个基序标签，C-末端具有MEEVD基序，属于胞质型热激蛋白。系统发育分析显示，Bc-Hsp90基因高度保守。不同高温（32-40 ℃）胁迫处理1 h和2 h后，瓜实蝇成虫体内Bc-Hsp90的相对表达量均显著高于对照组（26 ℃），并在38 ℃和40 ℃表达量最高；阿维菌素长期筛选的RS品系，Bc-Hsp90表达量是对照SS品系的2.13倍，但RS品系以LC90剂量诱导后，Bc-Hsp90表达量显著下调，随着恢复时间延长其表达量逐渐升高，96 h后恢复到对照水平。【结论】 推测Bc-Hsp90在瓜实蝇耐热性和抗阿维菌素过程中可能起着重要作用。
英文摘要：
[Objectives] To investigate the function of
the heat shock protein Hsp90 gene of Bactrocera cucurbitae (Coquillett) (Bc-Hsp90) under different temperatures
and pesticide stress in order to improve control of this pest. [Methods] The cDNA full-length fragment of the Bc-Hsp90 was cloned
using the RACE-PCR method. Bioinformatics tools were used to analyze the
sequence characteristics, and real-time quantitative PCR was used to detect the
expression levels of the gene under heat stress, and after exposure to the
pesticide abamectin, in abamectin-resistant and susceptible strains. [Results] The complete sequence of 2 654 bp was
obtained (Accession number: KP864677) and was found to contain a 2 145 bp open
reading frame (ORF) encoding 715 amino acids. Family signatures of the HSP90
protein and the cytoplasm of the Hsp90 gene C-terminal region MEEVD motif were detected. Phylogenetic analysis
indicates that Bc-Hsp90 is highly
conserved. Real-time quantitative PCR results showed that 1 h and 2 h of heat
stress（32-40℃）up-regulated the
expression of Bc-Hsp90. Expression
peaked at 38℃ and 40℃ and expression in an abamectin
resistant strain (RS) was 2.13 times higher than in a sensitive strain (SS).
The expression of the Bc-Hsp90 gene
was down-regulated after exposure to an LC90 dose of abamectin and returned to normal levels after 96 h. [Conclusion] The expression level of Bc-Hsp90 could play an important role in
heat resistance and pesticide tolerance in B.
cucurbitae.